The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
The proteins mediating RNA interference (RNAi) are part of an evolutionarily conserved cellular pathway that processes endogenous cellular RNAs to silence developmentally important genes (Grishok et al., 2001; Hutvagner et al., 2001). RNAi is a potent inhibitor of targeted gene expression in a wide variety of organisms (Wianny and Zernica-Goetz, 2000; Kennerdell and Carthew, 1998; Fire et al., 1998). The triggers for RNAi are small interfering RNAs (siRNAs) that are processed from long double stranded or hairpin RNA precursors and become part of a ribonucleoprotein complex (Lipardi et al., 2001; Sijen et al., 2001). RNAi mediated gene silencing in mammalian cells has been achieved by either transfecting synthetic dsRNA (Caplan et al., 2001; Elbashir et al., 2001), plasmids (Brummelkamp et al., 2002; Lee et al., 2002; Miyagishi and Taira, 2002) expressing siRNA as individual sense and antisense strands, or the use of short hairpin RNAs (shRNAs) 21-29 nucleotides long which are processed by the RNAse III family member Dicer to siRNA sized molecules that guide the cleavage of cognate mRNA (Xia et al., 2002; An et al., 2003). Most expression systems produce shRNAs from Pol III promoters which provide discrete 5′ and 3′ termini that are further processed into siRNAs. These transcripts mimic micro RNA precursors in structure and are most probably cytoplasmically exported by the transport factor exporting 5 (Ye et al., 2003).
Early on it was demonstrated that a Pol II promoter could also be used to express shRNAs (Xia et al., 2002). Some of the restrictions for expression of a functional shRNA include elimination of non-base paired 5′ sequences and the use of a minimal poly A signal (Xia et al., 2002). There are several potential advantages to utilizing Pol II expressed shRNAs including tissue specific promoters and inducible transcription. Given the requirement for a poly A signal at the 3′ end of the transcriptional units, it is not known how the final shRNAs are produced, and which transport system is used to export these to the cytoplasm. Nevertheless, it is clear that functional siRNAs can be produced by Pol II systems.
Small interfering RNAs (siRNAs) can induce potent gene silencing by directing degradation of cognate mRNAs. Controlled expression of siRNA may be important in clinical settings because it has been reported that some siRNA and shRNA sequences can induce a non-specific interferon response. The present invention thus provides controlled expression of siRNA.